小麦TaCBL1基因的克隆与功能分析

发布时间：2018-06-25 01:17

  本文选题：小麦 + CBL-CIPK　； 参考：《海南大学》2017年硕士论文

【摘要】：在自然环境中,植物经常受到多种胁迫刺激。这些刺激可以被植物感应并通过多种信号转导途径来产生不同形式的响应。钙(Ca2+)信号传导就是在植物中转导大量刺激或信号的一个非常重要的途径。Ca2+通过参与调节各类钙解码器以介导植物中的信号传导。在这些解码器中,钙调磷酸酶蛋白(CalcineurinB-likeproteins,CBL)和与CBL蛋白特异性互作的CIPK蛋白(CBL-interacting protein kinase)。这种激酶蛋白形成复合物并在转导这些信号中发挥非常重要的作用。这两个基因家族参与了多种刺激-反应耦合的信号网络通路。尽管CBL-CIPK网络已经证明能够在植物发育和各种环境胁迫的反应中起关键作用,但是在小麦中所起到的功能了解甚少。为了解小麦CBL1所起的作用,首先克隆出TaCBL1基因,对其编码的蛋白进行生物信息学分析。利用qRT-PCR技术分析TaCBL1基因在不同非生物胁迫下的表达模式,通过农杆菌介导法将TaCBL1基因转入烟草中,观察其对非生物胁迫的响应,测定其形态指标、生理生化指标等,揭示了过表达TaCBL1在非生物胁迫高盐处理下对烟草的生长发育的影响,主要的研究结果如下:1.通过对小麦进行不同的非生物胁迫(盐,低温,干旱,ABA)处理结果表明,TaCBL1基因在 PEG-6000(20%)、高盐(200 mM NaCl),低温(4℃)和 ABA(100μuM)处理的情况下,其表达量会随着时间的延长发生变化,其中TaCBL1基因在PEG模拟干旱处理时,发现3h时根中TaCBL1基因显著下降,之后慢慢升高,叶片中该基因表达在9h达到最低;200 mM盐处理下TaCBL1的表达水平一直较低,12h会出现瞬时性的增加之后又降低到低水平;低温(4℃)处理时,叶片中:TaaCBL/基因的表达量都较低,根中的表达量会有波动,但总体仍是低于Oh的表达量;ABA处理时,TaCBL1基因在叶片和根中的表达量都是逐渐降低的,且根中该基因的表达下降的更快。2.将TaCBL1基因构建到pCAMB1A1300载体上,在烟草原生质体中表达TaCBL1蛋白做定位分析,表明TaCBL1蛋白定位在细胞膜上,表明TaCBL1主要在质膜上起作用。3.利用农杆菌介导法将TaCBL1基因转入烟草中在不同的非生物胁迫下观察其表型,有趣的是其表现为对盐是敏感的,这与前人在拟南芥上AtCBL1表现出对盐的抗逆性不同,当然我们了解到前人在对杨树PeCBL1基因进行研究时也有类似的表型。4.为了进一步探究TaCBL1沿转入烟草为何会出现这种表型,我们取转基因烟草在不同浓度盐处理下的叶片和根部组织,做生理生化分析和钠钾离子含量的测定,结果表明盐处理下烟草叶和根中的K+/Na+,野生型都要比转基因烟草的高。
[Abstract]:In the natural environment, plants are often stimulated by a variety of stresses. These stimuli can be induced by plants and through multiple signal transduction pathways to produce different forms of response. Calcium (Ca 2) signal transduction is a very important pathway that transduces a large number of stimuli or signals in plants. [Ca 2] 2 participates in the regulation of various calcium decoders to mediate the signal transduction in plants. In these decoders, the CalcineurinB-like proteinsl and the CBL-interacting protein kinase). This kinase protein forms a complex and plays a very important role in transduction of these signals. These two gene families are involved in multiple stimuli-response coupled signaling network pathways. Although CBL-CIPK networks have been shown to play a key role in plant development and responses to various environmental stresses, little is known about the functions played in wheat. In order to understand the role of CBL1 in wheat, TaCBL1 gene was cloned and its encoded protein was analyzed by bioinformatics. The expression patterns of TaCBL1 gene under different abiotic stress were analyzed by qRT-PCR. TaCBL1 gene was transferred into tobacco by Agrobacterium tumefaciens. The response of TaCBL1 gene to abiotic stress was observed and its morphological indexes, physiological and biochemical indexes were measured. The effects of overexpression of TaCBL1 on the growth and development of tobacco under abiotic stress and high salt stress were revealed. The main results were as follows: 1. The results of different abiotic stress (salt, low temperature, drought abscisic acid) on wheat showed that the expression of TaCBL1 gene changed with time under PEG-6000 (20%), high salt (200mm NaCl), low temperature (4 鈩，

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